1. Field of the Invention
The present invention relates to oilseed crops producing valuable seeds having altered amino acid composition and fatty acid composition, and to a method for preparing such crops. More specifically, the present invention relates to oilseed crops with an introduced antisense gene of seed storage protein and to a method for preparing such crops.
2. Description of Related Art
The term "oilseed crops" as used herein refers to crops which are used to obtain oils (fats) from their seeds. Such crops have been widely cultivated as sources for edible oils, such as a rapeseed oil and a sesame oil, or as sources for a variety of oils for industrial use. For example, Brassica plants, of which seeds include lipids at about 60% of the seed weight, are cultivated in various places in the world. The oilseed meals contain proteins at high level and have been used as feed and fertilizers.
However, a rapeseed oil has been known to contain an erucic acid, which is harmful to human, and oilmeal of Brassica species has been known to contain glycocynolates which are associated with toxic effects on livestocks. Therefore, a great effort had been made to lower the amount of such harmful components by conventional plant breeding. As the result, a "double low" variant has been developed in Canada, which contains erucic acid at less than 2% of the seed weight in its seed oil, and glycocynolate at less than 30 micromol per 1 g of meal.
Recently, oil consumption has increased and the associated market has become in need of diversity of oil. Accordingly, oilseeds with high lipid content and with valuable fatty acid compositions are needed for a variety of purposes. For example, as edible oil, oilseeds containing negligible erucic acid and low levels of saturated fatty acid are desired because such oils are good for human health. On the other hand, oilseeds containing large amounts of erucic acid, medium-chain fatty acid, and/or polyunsaturated fatty acid are desired for industrial purpose. As for meal, those including a large amount of proteins or essential amino acids are desired.
It is laborious to develop new desirous breeds and it is difficult to alter valuable components in seeds for specific purpose by conventional breeding methods with crossing. Conventional breeding with crossing comprises very laborious and time-consuming processes which aim at the selection of desired breeds from various variants and establishment of pure lines. Other methods, such as .gamma.-ray irradiation and somaclonal variation, have been conducted in an attempt of obtaining desired breeds from various variants. However, breeds obtained by these methods often cannot be used for cultivation because, in addition to targeted genes, other genes in breeds are often mutated simultaneously.
On the contrary, methods involving gene engineering technique are advantageous for specifically preparing desired breeds, because such methods permit the alteration of a specific gene alone and introduction of the targeted gene into crops. In more detail, such methods comprise the steps of 1) isolating a gene encoding desired phenotype, 2) modifying the gene so that the gene is expressed in desired tissues or sites, and 3) introducing the gene into the crops to express the desired phenotype.
Examples of genes encoding targeted phenotype in the above step 1) include genes encoding enzymes involved in biosynthesis of seed storage compounds and genes of storage proteins. Seed storage comounds are essentially lipids, proteins, and carbohydrates, which vary in amount depending on plants. It is known that these compounds are accumulated during embryogenesis, and that the biosynthesis pathways of these compounds are closely related. In more detail, these compounds are synthesized from the same and identical starting substance. As for Brassica species, it is known that lipids and proteins are accumulated in seeds, but all enzymes involved in biosynthesis of storage lipids are not isolated. Genes for napin or cruciferin, which are storage proteins, have been isolated in Arabidopsis (Plant Phys., 87, 859-866, 1988; Plant Mol. Biol., 11, 805-820, 1988), Raphanus sativus (Plant Mol. Biol., 20, 467-479, 1992; Gene, 99, 77-85, 1991), Brassica napus (Plant Mol. Biol., 5, 191-201, 1985; Plant Mol. Biol., 14, 633-635, 1990), and the like.
The above step 2) is selection and construction of "DNA part", which is an antisense gene (antisense oligonucleotides) of a given storage protein and suppresses the expression of the gene and alters the phenotype of a crop, when introduced into the crop. As for B. napus, an example of expression of a chimeric gene consisting of DHFR gene and napin gene driven by the napin promoter has been reported (S. E. Radde, Theor. Appl. Genet., 75, 685-694, 1988), but no example of introduction of an antisense gene of a storage protein has been reported. As for introduction of antisense gene, there is a report that when antisense gene of ADP-glucose-pyrophosphorylase, one of the starch synthesizing enzymes in potato, has been introduced into potato, the amount of starch was decreased and the amount of sucrose and certain proteins were increased (EMBO J., 11, 1229-1238, 1992).
The above step 3) is a process for introducing a desired gene into a crop. As for B. napus, for example, there is known a method for regenerating plants from protoplasts introduced with the targeted DNA by electroporation (Plant Science, 52, 111-116, 1987). A method for regenerating a plant introduced with targeted DNA by Agrobacterium-mediated transformation is also known (Japanese Laid-Open Patent Publication No. 1-500718).